TRAIL-Induced Keratinocyte Differentiation Requires Caspase Activation and p63 Expression  Nan-Lin Wu, Te-An Lee, Te-Lung Tsai, Wan-Wan Lin  Journal of Investigative Dermatology  Volume 131, Issue 4, Pages 874-883 (April 2011) DOI: 10.1038/jid.2010.402 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Induction of differentiation markers in NHEKs by TRAIL. (a) NHEKs were treated for 30hours with TRAIL and assayed for the expression of differentiation markers by immunoblotting. (b) NHEKs were treated with TRAIL (300ngml-1) for different periods. Expression of TGase 1, involucrin, keratin 10, loricrin, and profilaggrin was determined. (c) Under different periods of culture in a high-calcium medium (1.5mM), the expression of TGase 1, involucrin, and the receptors of TRAIL, including DR4, DR5, DcR1, and DcR2, was assayed by immunoblotting. (d) Cultured NHEKs were treated with TRAIL (300ngml-1) for 30hours. Immunofluorescence staining was performed to detect the expression of involucrin and TGase 1 (green color). Nuclei were visualized by DAPI (blue). DAPI, 4′,6-diamidino-2-phenylindole; DcR, decoy receptor; DR, death receptor; NHEK, normal human epidermal keratinocyte; TGase, transglutaminase; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand. Bar=50μm. Journal of Investigative Dermatology 2011 131, 874-883DOI: (10.1038/jid.2010.402) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 TRAIL induces the mRNA expression of differentiation markers in NHEKs. (a) Under the treatment of TRAIL at different concentrations for 24hours, the changes of TGase 1 and involucrin mRNA in NHEKs were assayed. (b) After treatment with TRAIL (300ngml-1), high-calcium medium (1.5mM), or PMA (50nM), the time course of TGase 1 mRNA change was evaluated. Similar to panel b, time-dependent mRNA changes of (c) involucrin, (d) keratin 10, (e) loricrin, and (f) profilaggrin were also examined. Real-time quantitative PCR analysis was performed to determine the mRNA changes of differentiation markers. Student’s t-test was performed comparing the relative mRNA expression of those differentiation markers with untreated control cells. *P<0.05 (mean±SEM n=3). NHEK, normal human epidermal keratinocyte; PMA, phorbol 12-myristate 13-acetate; TGase, transglutaminase; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand. Journal of Investigative Dermatology 2011 131, 874-883DOI: (10.1038/jid.2010.402) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 TRAIL-induced apoptosis and differentiation are mediated by caspase activation. (a) NHEKs were treated with TRAIL for 2.5hours. The cell lysates were subjected to immunoblotting to detect the cleavage of procaspases 3 and 8. Under the pretreatment of zDEVD-fmk (10μM) or zIETD-fmk (10μM) for 30minutes, (b) the cell death of NHEKs treated with TRAIL for 24hours was measured by the MTT assay (*P<0.05, n=3), (c) the cleavage of procaspases 3 and 8 in NHEKs with the treatment of TRAIL for 2.5hours was evaluated by immunoblotting, and (d) NHEKs treated with TRAIL (upper panel), 1.5mM CaCl2, or PMA (50nM) (lower panel) for 30hours were assayed for the expression of TGase 1 and involucrin by immunoblotting. MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-dephenyltetrazolium bromide; NHEK, normal human epidermal keratinocyte; PMA, phorbol 12-myristate 13-acetate; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand. Journal of Investigative Dermatology 2011 131, 874-883DOI: (10.1038/jid.2010.402) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Differential regulation of MAPK signaling by TRAIL in NHEKs. (a) NHEKs were treated with TRAIL (300ngml-1) for the indicated time points. Immunoblotting with specific antibody against ERK1/2 or p38 was used to assess the activation of MAPK signaling. (b) Increasing the period of treatment, the change of phosphorylated ERK1/2 and p38 elicited by TRAIL was also evaluated. (c) Under the pretreatment of zDEVD-fmk (10μM), zIETD-fmk (10μM), or zVAD-fmk (10μM), NHEKs were treated with TRAIL for 3hours. Cell lysates were then assayed by immunoblotting to evaluate the effects of caspase inhibition on the TRAIL-induced signaling of ERK1/2 and p38. (d) Pretreatment of SB203580 (3μM) was performed to examine the roles of p38 in TRAIL-induced keratinocyte differentiation. ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; NHEK, normal human epidermal keratinocyte; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand. Journal of Investigative Dermatology 2011 131, 874-883DOI: (10.1038/jid.2010.402) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 TRAIL induces the degradation of p63 by activation of caspase. (a) NHEKs were treated with TRAIL for 24hours. The expression of p63 was evaluated by immunoblotting. (b) Real-time quantitative PCR analysis was used to investigate the mRNA change of ΔNp63 isoforms in NHEKs treated with TRAIL. (c) NHEKs were cultured in high-calcium medium (1.5mM), or treated with TRAIL (300ngml-1) for the indicated periods. The change of p63 expression was detected by immunoblotting. (d) NHEKs were treated with cycloheximide (CHX) (10μgml-1), TRAIL (300ngml-1), and/or caspase inhibitors for the indicated periods. Data of p63 immunoblotting were expressed as a percentage of control protein expression. *P<0.05 versus TRAIL+CHX (mean±SEM n=3). NHEK, normal human epidermal keratinocyte; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand. Journal of Investigative Dermatology 2011 131, 874-883DOI: (10.1038/jid.2010.402) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Knockdown of ΔNp63 decreases TRAIL-induced differentiation but barely affects TRAIL-induced cell death. (a) Real-time quantitative PCR and immunoblotting confirmed the knockdown of ΔNp63. (b) The MTT assay determined the survival of NHEKs with ΔNp63 siRNA for 72hours and (c) NHEKs with TRAIL treatment for an additional 24hours after knockdown of ΔNp63 for 48hours. (d) Immunoblotting examined the effect of ΔNp63 siRNA on the TRAIL-elicited cleavage of procaspase 3. (e) The effects of ΔNp63 siRNA on the TRAIL-induced expression of mRNA and protein of involucrin and TGase 1. (f) MAPK signaling in NHEKs treated with TRAIL (300ngml-1) after knockdown of ΔNp63 for 48hours *P<0.05 versus scramble siRNA (mean±SEM n=3). MAPK, mitogen-activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-dephenyltetrazolium bromide; NHEK, normal human epidermal keratinocyte; siRNA, small-interfering RNA; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand. Journal of Investigative Dermatology 2011 131, 874-883DOI: (10.1038/jid.2010.402) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions